The long term objective of this research program is to significantly enhance our understanding of megakaryopoeisis at the molecular level. Three specific aims are proposed. The first is to document the role of the c-ski proto-oncogene in megakaryopoeisis. Preliminary studies in several human hematopoietic tumor cell lines showed that expression of the c-ski proto-oncogene is specifically induced during in vitro megakaryocyte differentiation but not during erythroid or myeloid differentiation. The importance of ski expression in mediating megakaryocyte differentiation will be tested by inhibiting its activity in various hematopoietic cell lines through use of antisense oligonucleotides, antisense RNA, ribozymes, and a dominant-negative mutant of ski. Additionally, overexpression of ski in the same cell lines will be examined to determine if ski expression, by itself, is sufficient to induce the initial stages of megakaryocytic differentiation. The ski protein appears to be a transcription factor that binds specific responsive elements in DNA, and these sequences have been located in the promoter regions of four platelet/megakaryocytic specific genes, as well as in the upstream region of the gene encoding the erythroid/megakaryocytic transcription factor GATAI. Therefore, the role of ski-responsive elements in these cell lines will also be examined, through the use of appropriate reporter constructs. The second specific aim is to identify, in a model cell line, CHRF-288-11, mRNAs whose expression changes during megakaryocyte differentiation. These will be identified using two recently developed PCR-based cDNA cloning techniques; differential display, and a gene expression screen. Once obtained, these cDNAs will be used for the experiments described in the third specific aim, which is to determine if their expression is necessary and/or sufficient for differentiation to occur. These experiments will employ overexpression and inhibition of expression techniques similar to the ones outlined in the first specific aim, in which the role of the c-ski protooncogene was assessed. In this manner genes important in regulating megakaryopoeisis will be identified allowing their mechanism(s) of action to be determined. With this information in hand, it may become feasible to design agents to artificially manipulate the expression of these genes, which has important implications both for treating diseases in which platelet production is impaired, and for engraftment of megakaryocytes after bone marrow transplantation.